Abstract For many years, animal models have been greatly useful for dissecting basic mechanisms of allograft rejection and tolerance. However, the translation of basic studies to clinical intervention has been an arduous challenge. The majority of published small animal allograft studies utilize relatively young healthy animals, both as transplant donors and recipients. One can argue that these models often do not reflect several features of clinical transplantation that can impair the tolerance process. In response to this dilemma, many more recent studies have focused on identifying key rate-limiting, clinically relevant obstacles to allograft tolerance induction. This proposal focuses on identifying how a particular route of immune memory results in the disruption of tolerance in a mouse model of islet transplantation. The prevailing view of immune memory as a barrier to tolerance is that prior exposure to environmental antigens, pathogens, and vaccination antigens generates a burden of antigen-experienced T and B cells that can spontaneously cross react to allogeneic MHC molecules. However, our recently published studies indicate that vaccine-induced memory with little if any cross-reactivity to donor MHC can nevertheless disrupt tolerance if the donor cells express the vaccine-directed antigen. We refer to this type of reactivity as 'incognito' immune memory simply because it is not readily detected by pre-transplant host monitoring for anti-donor MHC reactivity. As such, this scenario models a common transplant setting in which the donor graft harbors non-MHC antigens that can be recognized by host immunity generated through prior exposure to pathogens, vaccinations, or pre-existing autoimmunity. We posit that this type of common host immunity can be disregarded and yet it can readily impair tolerance induction without any requirement for cross-reactivity to donor MHC antigens. This project will determine the mechanisms of how this form of immune memory disrupts tolerance by addressing the general working model: Non-MHC-directed memory can disrupt tolerance by simultaneous 'linked' recognition of alloantigens and vaccine- or virus-induced antigen specific memory in vivo. Implications of this overall model will be addressed through the following three Specific Aims: Specific Aim 1: Determine the conditions of memory-directed antigen expression required to disrupt tolerance. Specific Aim 2: Determine the impact of memory cells on the activation of nave, graft reactive T cells. Specific Aim 3: Determine the impact of physiologically relevant host and donor gammaherpesvirus 68 (gHV68) infection on the subsequent capacity to induce transplant tolerance. Taken together, the goal of this project is to dissect how this alternative form of immune memory disrupts tolerance. We propose that understanding how such 'incognito' memory impacts tolerance will identify key target pathways for future intervention and potentially expand how transplant candidates/hosts/recipients are screened for anti-donor immune reactivity prior to transplant.